Vitis 45 (1), 29–36 (2006)

Assessment of resistance of and necator pathogenicity using a laboratory assay

J. P. PÉROS1), T. H. NGUYEN2), C. TROULET2), C. MICHEL-ROMITI2) and J. L. NOTTEGHEM2)

1) UMR DGPC and 2) UMR BGPI, INRA, Montpellier, France

Summary sources of resistance and to assist breeding programs for cultivars being resistant to powdery mildew. Determination To develop a quantitative evaluation of grapevine resist- of host resistance was based on field observations (BOUBALS ance to powdery mildew and of pathogenicity of the causal 1961, DOSTER and SCHNATHORST 1985 a, STAUDT 1997), tests agent (Erysiphe necator), a spot inoculation method was on leaf disks (STEIN et al. 1985, WANG et al. 1995) or on in developed using detached leaves of potted plants. The per- vitro material (KITAO and DOAZAN 1990). The results obtained centage of inoculating spots leading to a colony and the by different methods were found to be correlated (LI 1993). mean diameter of colonies were determined to assess the However, data were mostly presented on discrete scales of host-pathogen relationships. Significant differences were disease rating. Continuous variables would be more appro- found between host cultivars and their ranking was associ- priate for quantitative analysis of the relationships between ated with that observed in the . There was a signifi- grapevine and E. necator. cant interaction between cultivar and replicate during the Moreover, quantitative assessment of the pathogenic- whole experiment indicating that the physiological state of ity of E. necator would enable to choose the most appropri- detached leaves is important. Aging of tissues was accom- ate isolates to rate host genotypes. The diversity of the panied by a gain in resistance that was considerably more pathogen with respect to its pathogenicity has been poorly marked in resistant cultivars. Only partly expanded leaves investigated. KITAO and DOAZAN (1990) reported differences of resistant cultivars that stopped expansion on the agar in colony diameter, length of latent period and production of medium supported the development of the . Signifi- conidia between a few isolates from different European re- cant differences between E. necator isolates were also dem- gions. Some evidence for pathogenic specialization was onstrated, but these variations were less marked than those observed when pathogenicity was examined for isolates due to host cultivar and leaf position. Preliminary results obtained from a range of Parthenocissus and obtained with different isolates from the two European ge- (GADOURY and PEARSON 1991). A quantitative method in a netic groups (A and B) indicate that, on average, group A is closed-system laboratory environment would thus be par- less pathogenic. ticularly useful to address several questions regarding the diversity of the pathogen. For instance, the marked genetic K e y w o r d s : powdery mildew, , Vitis hybrids, differences between the morphologically similar groups that rating method, ontogenic resistance, Erysiphe necator, have been identified in European, Indian and Australian necator, pathogenicity. populations of E. necator (DÉLYE et al. 1997, STUMMER et al. 2000) suggest that these groups may differ in pathogenicity, Introduction but this possibility has not been explored. The objectives of this study were 1) to develop a method Powdery mildew caused by Erysiphe necator Schwein. to obtain a simple, quantitative assessment of the interac- (formally (Schwein.) Burrill (BRAUN and tion between grapevine and E. necator and 2) to evaluate TAKAMATSU 2000) is an economically important disease of the potential of this method using a set of grapevine grapevine (BULIT and LAFON 1978, PEARSON 1988). Several cultivars with different levels of susceptibility and resist- asexual cycles of reproduction of the fungus occur during ance in the vineyard as well as a set of isolates differing in the growing season and may cause losses of and qual- host origin and genetic group. ity (GADOURY et al. 2001, CALONNEC et al. 2004, STUMMER et al. 2005) if there is no or incomplete chemical control. The most efficient way to minimize powdery mildew is Material and Methods to grow resistant cultivars, however, the elite cultivars origi- nate from Vitis vinifera which is the most susceptible host P l a n t m a t e r i a l : Four Vitis vinifera cultivars species. Monogenic resistance to E. necator has been stud- (Cabernet-Sauvignon, Carignan, Cinsault, Grenache) and ied in Muscadinia and its hybrids with Vitis vinifera (BOU- three resistant interspecific cvs (Villard Blanc, Villard Noir, QUET 1986, PAUQUET et al. 2001). In the Vitis resist- Jacquez) were used. Villard Blanc and Villard Noir have a ance appears to be due to several genes (BOUBALS 1961, complex genealogy involving several Vitis species with a FISHER et al. 2004). Methods are needed to identify valuable majority of V. rupestris and V. vinifera parentage (ANTCLIFF

Correspondance to: Dr. J. P. PÉROS, UMR DGPC, INRA Equipe Génétique Vigne, 2 place Viala, 34060 Montpellier cedex, France. Fax: +33-4-9961-2064. E-mail: [email protected] 30 J. P. PÉROS et al.

1992). Jacquez probably results from a cross between leaves every 4-5 weeks under the conditions described V. aestivalis and V. vinifera (GALET 1988). For this study, above. plants were obtained from one-bud cuttings prepared in P a t h o g e n i c i t y a s s a y : Inoculum was produced spring from one-year old branches collected in the preced- on detached leaves of cv. Cabernet-Sauvignon inoculated ing winter and stored at 4 °C. Cuttings were placed at high with infected tissues and incubated for 2-3 weeks under the density in flat boxes filled with vermiculite and maintained conditions described above. Detached leaves of potted for 6-8 weeks in a growth chamber at 25 °C in the light plants were inoculated using a spot method. About 20-60 (12 h·d-1, 40 µmol quanta m-2 s-1) and 22 °C in the dark. Rooted conidia were taken from a leaf infected with powdery mildew cuttings were then transferred in 2 liter pots containing a using a glass needle. Inoculum was deposited on a healthy mixture of soil and vermiculite (2:1), fertilized (Osmocote, leaf by gentle contact with the leaf epidermis. On each in- Scotts Europe B.V., Heerlen, The Netherlands) and grown in oculated leaf, three separate spots were applied approxi- the greenhouse. Plants were regularly trimmed to allow the mately in the middle between the petiole insertion point and development of new shoots with suitable leaves. the lobe extremity, avoiding veins. Inoculated leaves were Leaves of potted plants were removed, washed in tap incubated under the conditions described above. Inocula- water and disinfected for 1.5 min in a 0.3 % solution of active tion was considered successful if the spot resulted in a colony chloride. After three rinses in sterile distilled water, leaves that sporulated. The diameter of the area that sporulated were dried between several layers of sterile paper. Petiole was measured under the binocular microscope 11 d (Experi- length was then reduced to 1.5-2 cm. Leaves with their up- ment 3) or 20 d (other experiments) after inoculation. per surface visible were placed on 8 g·l-1 agar medium in 9 cm E x p e r i m e n t a l d e s i g n s a n d a n a l y s e s : Five Petri dishes; the remaining part of the petiole was inserted different experiments were performed using complete block into the medium. designs (Tab. 2). Each experiment was replicated three times F u n g a l i s o l a t e s : The isolates of E. necator used and, depending on the experiment, the replicate was studied in this study originated from infected material collected in as a second or a third classifying factor. Isolate FA01 was southern France (Ardeche district) in 2000 and 2001. In this used in experiments 1 and 2. Cv. Cabernet-Sauvignon was region, a population study identified the two genetic groups used in experiment 4. Leaves were taken at 5 different posi- that exist in Europe (PÉROS et al. 2005), and the set of isolates tions: the first expanding leaf (position 1), the second ex- included 5 group A isolates and 4 group B isolates (Tab. 1). panding leaf (position 2), and the three following leaves Within the two d after collection in the field, infected tissues (positions 3-5). The different positions were compared in were brushed onto detached leaves of cv. Cabernet- experiment 2 and position 2 and 4 were compared in experi- Sauvignon. The inoculated leaves were then incubated for ment 4. The leaves used in experiment 1 and 5 were taken at 10-15 d at 25 °C under 16·h d-1 illumination (40 µmol quanta position 2 and those used in experiment 3 were taken at m-2 s-1). Either a single or a few conidia from the same co- position 1. Statistical analyses were performed using SAS nidial chain were then picked up using a glass needle under (version 8.1; SAS Institute, Cary, NC, USA). The numbers of the binocular microscope. Conidia were spotted on detached colonies that sporulated were compared with χ2 testing us- leaves from the greenhouse or on leaves of in vitro plants, ing the FREQ procedure. Analyses of variance for the diam- cv. Cinsault, cultured as described by PÉROS et al. (1998). eter of the colony were performed using the GLM procedure The process was repeated once. The isolates were main- in SAS and means were compared using paired t-tests or the tained on leaves of in vitro plants and subcultured on new CONTRAST statement.

T a b l e 1

Origin of Erysiphe necator isolates used in this study

Isolate Sampling date Tissues Host cultivar Genetic groupx (month/year)

FA01 07/2000 Leaf Carignan A FA03 07/2000 Berry Jacquez B FA09 07/2000 Leaf ndy A FA12 07/2000 Leaf Villard Noir A FA16 05/2001 Flagshoot Carignan A FA18 09/2001 Leaf nd B FA19 09/2001 Leaf nd B FA20 09/2001 Leaf nd B PA07 11/2001 Leaf Gamay B

x Assignment to genetic groups was based on results obtained using Nested allele specific PCR (DÉLYE et al. 1999), groups A and B corresponding to groups I and III described in Europe by DÉLYE et al. (1997). y Not determined. Powdery mildew resistance of grape and Erysiphe necator pathogenicity 31

T a b l e 2

Experiments designed to evaluate the performance of a spot inoculation method of grapevine with Erysiphe necator

Exp. Source of variation (levels) Blocks Exp. Unit Dates of replicates

1 Cultivar (CAB, CAR, CIN, GRE, VB, VN, JAC)x 5 4 leaves 21/05, 04/06 and 17/06/02 2 Cultivar (CAB, GRE, VN) Leaf position (n°1 to n°5)y 5 2 leaves 21/08, 29/08 and 10/09/02 3 Cultivar (CAB, VN, JAC) Isolate (FA01, FA03, FA12)z 3 2 leaves 03/10/02, 17/04 and 01/09/03 4 Isolate (FA01, FA09, FA12, FA16, FA18, FA19, FA20, PA07) Leaf position (n°2 and n°4) 3 2 leaves 04/07, 17/07 and 14/08/03 5 Cultivar (CAB, GRE, VN) Isolate (FA01, FA16, FA18, FA20) 3 2 leaves 10/07, 20/08 and 26/08/03 x Cultivars were coded as follows: CAB: Cabernet-Sauvignon, CAR: Carignan, CIN: Cinsault, GRE: Grenache, VB: Villard Blanc, VN: Villard Noir, JAC: Jacquez. y The leaf at position 1 is the first expanding leaf. z Origin of isolates is given in Tab. 1.

Results cant effect of the replicate except in experiment 1 (Tab. 4). Large significant differences for colony diameter between O v e r a l l p e r f o r m a n c e o f t h e m e t h o d : replicates were evidenced in experiment 2, 3 and 5. The ef- During the course of this sudy a total of 1536 detached fect of replicate interacted with cultivar response in all ex- leaves were used and 4608 inoculating spots were made. periments in which different cultivars were tested and with The percentage of spots observable 20 d after inoculation leaf position in experiment 4, but not in experiment 2. Con- (11 d in experiment 3) was 97.7 % indicating a very low rate versely, the isolate-replicate interaction term was never sig- of missing data. A few leaves, mostly in experiments 1 and 2, nificant (Tab. 4). showed necrotic lesions or were contaminated by sapro- E f f e c t o f v i n e c u l t i v a r : In experiment 1, phytic fungi. A few inoculating spots were contaminated by inoculation of Jacquez was not followed by development of other fungi despite the axenic precautions taken during the the fungus whatever the replicate (Tab. 5). The other preparation of inoculum and during inoculation. Leaves at cultivars fell into different groups: Cinsault and Carignan position 2 of the cv. Cabernet-Sauvignon inoculated with with maximal percentage in the three replicates, Cabernet- isolate FA01 corresponded to the most frequent combina- Sauvignon, Grenache and Villard Blanc with high percent- tion tested (experiment 1, 2, 4 and 5) and colony develop- ages but with variation between replicates, and Villard Noir ment was observed in 87 % of the 378 observable spots. with lower percentages. These results were confirmed for E f f e c t o f r e p l i c a t e : The percentage of infection the cultivars used in several experiments at leaf position varied in each experiment as a function of the replicate no. 2: the percentage of infection was 92 %, 84 % and 83 % (Tab. 3). In each experiment, χ2 testing showed significant for Cabernet-Sauvignon in experiment 2, 4 and 5, 83 % and differences between replicates. Generally one replicate was 90 % for Grenache in experiment 2 and 5, and 73 % and 51 % clearly distinguished from the two other replicates. Analy- for Villard Noir in experiment 2 and 5. In experiment 3, using sis of variance for colony diameter also revealed a signifi- leaves at position 1, some spots initiated infection in

T a b l e 3

The performance of a spot inoculation method of grapevine with Erysiphe necator: differences between replicates in each experiment

Experiment 1 Experiment 2 Experiment 3 Experiment 4 Experiment 5 Replicate % Ix D (mm)y % Ix D (mm)y % Ix D (mm)y % Ix D (mm)y % Ix D (mm)y

1 77a 12.2 49b 7.9a 65a 6.5a 91a 10.6b 71b 10.7a 2 71a 11.3 58a 8.0a 62a 4.3bc 71b 8.1a 82a 12.5a 3 63b 11.4 61a 11.6b 43b 5.1c 71b 10.0b 71b 11.1a x Percentage of infection based on the number of inoculating spots resulting in a colony. Percentage within the same column followed by the same letter are not significantly different at the P = 0.05 level of probability based on paired χ2 tests. y Mean diameter of sporulating colonies. Only data for inoculating spots resulting in a colony were included. Means within the same column followed by the same letter are not significantly different at the P = 0.05 level of probability based on paired t tests. ANOVA analyses for D are shown in Tab. 4. 32 J. P. PÉROS et al.

T a b l e 4

Analyses of variance for colony diameter after inoculation of detached leaves of grapevine with Erysiphe necator in five different experiments designed to study the effects of cultivar, isolate, leaf position and replicate

Experimentx Source of variationy df Mean square F value P

1 Cultivar 5 0.46 15.5 <0.001 Cultivar × Replicate 12 0.14 4.6 <0.001 Replicate 2 0.08 2.8 0.070 2 Replicate 2 2.29 47.2 <0.001 Leaf position 4 1.99 41.0 <0.001 Cultivar 2 0.78 16.1 <0.001 Cultivar × Replicate 4 0.12 2.5 <0.05 Replicate × Leaf position 8 0.06 1.3 0.25 Cultivar × Leaf position 8 0.04 0.8 0.57 Cultivar × Replicate × Leaf position 13 0.03 0.6 0.88 3z Cultivar 1 0.40 25.5 <0.001 Replicate 2 0.22 14.2 <0.001 Isolate 2 0.07 4.5 <0.05 Cultivar × Replicate 2 0.06 4.0 <0.05 Isolate × Replicate 4 0.03 2.0 0.12 Cultivar × Isolate 2 0.01 0.9 0.41 Cultivar × Isolate × Replicate 4 0.01 0.2 0.96 4 Leaf position 1 6.07 116.37 <0.001 Replicate 2 0.81 15.54 <0.001 Isolate 7 0.69 13.24 <0.001 Leaf position × Replicate 2 0.31 5.97 <0.01 Isolate × Leaf position 7 0.07 1.34 0.24 Isolate × Replicate 14 0.06 1.22 0.27 Isolate × Leaf position × Replicate 14 0.05 0.87 0.59 5 Cultivar 2 3.91 68.76 <0.001 Isolate 3 0.38 6.72 <0.001 Replicate 2 0.32 5.71 <0.01 Cultivar × Replicate 4 0.24 4.14 <0.01 Isolate × Cultivar 6 0.05 0.90 0.50 Isolate × Cultivar × Replicate 12 0.04 0.77 0.68 Isolate × Replicate 6 0.03 0.59 0.74

x Only data for inoculating spots leading to a powdery mildew colony were included. y In each experiment the sources of variation were ranked in decreasing order of F values. z Data for the Jacquez cultivar were not included in the analysis because a many values were lacking. cv. Jacquez (Fig. 3) and the average percentage of infection was 92 %, 67 % and 9 %, for Cabernet-Sauvignon, Villard Noir and Jacquez. The diameter of the powdery mildew colony also varied depending on the cultivar (Tab. 4). In experiment 1, the means per cultivar were compared for each replicate (Tab. 5). There were some changes in the ranking of cultivars between repli- cates as well as in the magnitude of differences between cultivars. In replicate 1 the cultivars were not separated, in replicate 2 only Villard Noir was distinguished from the other cultivars, and in replicate 3 the two hybrids were distin- guished from the V. vinifera cultivars. Cabernet-Sauvignon and Grenache were not separated in experiment 2 (Fig. 2) and experiment 5 (data not shown) whereas Villard Noir pre- sented colonies with smaller diameters than Cabernet- Sauvignon in experiment 2 (Fig. 2), experiment 3 (Fig. 3) and Fig. 1: Detached leaf of grapevine (cv. Carignan) 20 d after inocula- experiment 5 (data not shown). tion with Erysiphe necator using the spot inoculation method. Powdery mildew resistance of grape and Erysiphe necator pathogenicity 33

T a b l e 5

Percentage of infection and mean colony diameter for 7 grapevine cultivars inoculated with Erysiphe necator (isolate FA01) on detached leaves at position 2

Replicate 1 Replicate 2 Replicate 3 Cultivar % Ix D (mm)y % I D (mm) % I D (mm)

Cinsault 100a 13.4a 100a 12.8a 100a 14.5a Carignan 100a 13.1a 100a 12.7a 98a 12.1ab Cabernet-Sauvignon 97ab 11.9a 98a 13.8a 82b 13.7ab Grenache 93b 12.0a 68c 11.2a 77b 11.6bc Villard blanc 88b 11.6a 89b 11.5a 42c 6.1d Villard noir 58c 10.9a 40d 7.4b 28d 9.3c Jacquez 0 - 0 - 0 -

x Percentage of infection based on the number of inoculating spots resulting in a colony. Percentages within the same column followed by the same letter are not significantly different at the P = 0.05 level of probability based on paired χ2 tests. y Mean diameter of sporulating colonies. Only data for inoculating spots resulting in a colony were included. ANOVA analysis for D is shown in Tab. 4. Means within the same column followed by the same letter are not significantly different at the P = 0.05 level of probability based on t tests.

Fig. 2: Effects of cultivar and leaf position on percentage of infec- Fig. 3: Effects of cultivar and isolate of Erysiphe necator on per- tion and colony size 20 d after inoculation of detached leaves of centage of infection and colony size 11 d after inoculation of de- grapevine with isolate FA01 of Erysiphe necator, (experiment 3). tached leaves of grapevine (experiment 3). In the legend the code of Bars with a different letter for each cultivar were significantly each isolate is followed by the name of the cultivar from which it different at P = 0.05. was collected. For details see Fig. 2.

E f f e c t o f l e a f p o s i t i o n : In experiment 2, there the magnitude of the difference (84 % vs 72 %) was less was a dramatic decrease in the percentage of infection be- marked than in experiment 2 (92 % vs. 58 %). tween leaf position 2 and leaf position 3 (Fig. 2). This effect The diameter of the powdery mildew colony varied with was considerably more noticeable in Villard Noir than in the leaf position (Tab. 4, Fig. 2). The decrease was not linear: two V. vinifera cultivars. In Cabernet-Sauvignon inoculated leaves at positions 1 and 2 showed similarly high suscepti- in experiment 4, there was a significant difference between bility, except for Villard noir, and then the size of the colony the leaves taken at positions 2 and 4 (χ2 = 16.6, P < 0.001) but size decreased sharply and was almost the same at the three 34 J. P. PÉROS et al. other positions. For Villard Noir, means for leaves at posi- tions 3 to 5 were not subjected to t-tests because of limited data. In Cabernet-Sauvignon inoculated in experiment 4, there was a significant difference between leaves at position 2 and 4 (11.6 mm vs 7.4 mm), i.e. a bigger difference than that observed in experiment 2 (10.7 vs. 7.4 mm). E f f e c t o f t h e i s o l a t e : In the experiment investigating the pathogenicity of three isolates with re- spect to their host origin (experiment 3), we inoculated leaves at position 1 and some infections were observed on cultivar Jacquez (Fig. 3). No differences between isolates were evi- denced in Cabernet-Sauvignon, in contrast to the hybrids for which isolate FA12 originating from Villard Noir displayed lower percentages of infection. Data on colony diameter for Jacquez were not included in the variance analysis (Tab. 4). The cultivar-interaction term was not significant indicating that the ranking of Cabernet-Sauvignon and Villard Noir was not influenced by the isolate inoculated. Despite an overall significant effect of the isolate (Tab. 4), the means per iso- late did not show significant differences either for Cabernet- Sauvignon or for Villard Noir (Fig. 3). In experiment 4, designed to compare the pathogenicity of 8 isolates from two genetic groups on leaves of cv. Cabernet-Sauvignon, the percentage of infection varied and Fig. 4: Percentage of infection and colony size measured 20 d after isolate FA19 resulted in the lowest percentage of infection inoculation of detached leaves of Cabernet-Sauvignon taken at two (Fig. 4). On average, groups A and B did not differ signifi- leaf positions and inoculated with isolates of Erysiphe necator cantly (79 % vs. 76 %, P = 0.32). The analysis of variance from two genetic groups (experiment 4). Data for the two leaf performed for the diameter of powdery mildew colonies re- positions were combined. White and grey bars represent group A vealed an effect of the isolate (Tab. 4) and paired t identified and group B, respectively. For details see Fig. 2. isolate FA09 as the less pathogenic isolate (Fig. 4). We then compared the means of groups using the statement CON- TRAST in SAS. There was a significant difference between groups A and B (8.6 vs. 10.5 mm, P < 0.001). Thus, on aver- age, group A showed 18 % reduction in pathogenicity com- pared to group B. However, the data obtained with isolate FA09 from group A might have made a major contribution to this result. In experiment 5, we selected the two isolates from each group that had produced the largest diameters in experiment 4 and compared their pathogenicity using three cultivars. There were significant differences for the percentage of in- fection (Fig. 5) and, on average, groups A and B differed significantly (69 % vs. 81 %, P < 0.001). The analysis of variance performed for the diameter of powdery mildew colo- nies revealed an effect of the isolate (Tab. 4), however paired t-tests did not distinguish isolates (Fig. 5). Using CON- TRAST, a significant difference was observed between groups A and B (10.6 vs. 12.3 mm, P < 0.001), and, on average, group A showed 14 % less pathogenicity compared to group B. The isolate-cultivar interaction term was not sig- nificant (Tab. 4).

Discussion Fig. 5: Percentage of infection and colony size measured 20 d after inoculation of detached leaves of three cultivars of grapevine in- The method developed here allows the interaction be- oculated with isolates of Erysiphe necator from two genetic groups tween grapevine and E. necator to be studied under con- (experiment 5). Data for the three cultivars were combined. White trolled conditions. After the collection of leaves in the green- and grey bars represent group A and group B, respectively. For house all operations are performed in a controlled labora- details see Fig. 2. Powdery mildew resistance of grape and Erysiphe necator pathogenicity 35 tory setting that ensures reproducible conditions. The spot tissues from more resistant ones. In addition, we showed method allows dry conidia to be deposited on specific areas that the effect of leaf position was considerably more pro- of detached leaves. In very susceptible cultivars and in the nounced in the resistant hybrids than in the V. vinifera most susceptible tissues, the percentage of spots that led to cultivars. This rapid decrease in susceptibility with tissue sporulation was very high in different replicated experiments. age probably explains the lack of powdery mildew in vine- The diameter of the resulting colony provides another char- yards of resistant hybrids. Their youngest tissues can be acter that can easily be measured under the binocular micro- infected but the development of the fungus is generally ar- scope. Although the latency period of E. necator from in- rested during aging of tissues before sporulation occurs. In oculation to occurrence of conidiophores with mature co- contrast, the youngest leaves placed in Petri dishes stop nidia is 5-6 d, we performed observations only 20 d after expanding and are therefore artificially maintained in a sus- inoculation. This decision was made because: (1) prelimi- ceptible status. This also provides an explanation for the nary data indicated that measurements at 10 d gave similar results in experiment 1, i.e., the resistant hybrids were not results but with more variability and therefore less precision clearly distinguished from susceptible cultivars because the in the statistical analysis, (2) under the binocular microscope inoculation was performed in the expanding leaf at position measurement of larger colonies is easier, and (3) a few colo- 2. With leaves at position 3-5, the magnitude of differences nies in resistant genotypes took longer to develop and in- between susceptible and resistant cultivars increased. Data fection percentage can consequently be underestimated if presented here thus suggest that leaves at position 3 have observations are made earlier. However, when the smallest to be inoculated to clearly distinguish resistant from sus- leaves were inoculated it was easier to perform the measure- ceptible cultivars. On the other hand, the youngest leaves ments earlier and in experiment 3, we consequently meas- have to be used to compare resistant cultivars since very ured the colonies 11 d after inoculation. In contrast to the resistant hybrids like Jacquez only developed infection if two characters we measured for this study, more time and the first expanding leaf was inoculated. resources would be needed to estimate the length of the Our results confirm that age-related resistance, often incubation period after successive observations or to deter- called ‘ontogenic resistance’, is expressed in grapevine mine the number of conidia produced per colony. Further- leaves inoculated with E. necator. DOSTER and SCHNATHORST more, previous studies on grapevine powdery mildew (1985 b) hypothesized that a resistance factor inhibits pen- (BOUBALS 1961, COUTINHO and MARTINS 1985, KITAO and etration of E. necator in old-aged leaves and consequently DOAZAN 1990) demonstrated that characters like colony size, slows colony development. The fungus indeed needs to incubation time and spore production are correlated. repeatedly penetrate and form secondary haustoria to colo- Without treatments, cultivars of Vitis vinifera nize the tissue surface. Ontogenic resistance to powdery are affected by destructive epidemics. Some differences have mildew has been investigated more precisely in grape ber- been found among V. vinifera cultivars and Grenache was ries (FICKE et al. 2002, 2003, 2004; GADOURY et al. 2003). Only previously considered to be less susceptible than Cabernet- berries inoculated with E. necator within 3 weeks after bloom Sauvignon, Cinsault and Carignan (BOUBALS 1961, LI 1993). developed severe disease symptoms (FICKE et al. 2002). In This result was not corroborated in our study since Grenache resistant berries, the pathogen is stopped within 24 h after was not always distinguished from very susceptible arrival of the inoculum, i.e. after the formation of the cultivars. In contrast to V. vinifera cultivars, the three hy- appressorium but before it is able to form haustoria and brids are not subject to epidemics even in the absence of secondary hyphae. Investigations have shown that cuticle fungicide treatments. In experiment 1, the fungus did not thickness, formation of papilla, accumulation of phenolics develop on leaves of Jacquez but the two other resistant or pathogenesis-related proteins may only in part explain hybrids showed colonies that were not always significantly ontogenic resistance (FICKE et al. 2002, 2004). The most likely smaller than those observed on V. vinifera cultivars. Moreo- but still unknown mechanism may involve biochemical or ver, there was a significant interaction between cultivars ultrastructural modifications of the cuticle or cell wall (FICKE and replicates with variations in both, the rank of cultivars et al. 2004). As observed for leaves, resistant hybrids ex- and the magnitude of the differences between them. The pressed ontogenic resistance in berries earlier than cultivars three replicates of experiment 1 were completed in a two- of V. vinifera (FICKE et al. 2003). month period and a likely explanation was the difference in The method described in this paper is also suitable for the growing conditions of the plants that produced the analyzing the variation in pathogenicity within E. necator. leaves. CHELLIMI and MAROIS (1991) also explained the vari- However, the differences between isolates of E. necator were ability in their results by differences in the physiological less marked than the differences in tissue susceptibility due state of host tissues. to the cultivar or the leaf position. It would probably be Senescing leaves became more resistant to E. necator necessary to increase the number of replicates to measure and this effect was evidenced for both the success of infec- the differences between isolates more precisely. This method tion and the diameter of the colony. An effect of tissue age could also be used to check a range of host cultivars against on powdery mildew development has already been described a range of isolates from different hosts to analyze specializa- by DELP (1954) and DOSTER and SCHNATHORST (1985 b). How- tion in the fungus. The limited data presented here suggest ever, in contrast to the latter authors, we did not observe that isolates collected on resistant hybrids did not appear to that resistance increased linearly with an increase in leaf have developed a higher degree of pathogenicity. It is very maturity but that a threshold separated very susceptible likely that inoculum infecting resistant hybrids originated 36 J. P. PÉROS et al. from neighboring cultivars of V. vinifera where the epidemic DÉLYE, C.; RONCHI, V.; LAIGRET, F.; CORIO-COSTET, M. F.; 1999: Nested started. Two experiments indicated that, on average, group allele-specific PCR primers distinguish genetic groups of Uncinula necator. Appl. Environ. Microbiol. 65, 3950-3954. A displayed lower pathogenicity than group B. However, to DOSTER, M. A.; SCHNATHORST, W. C.; 1985 a: Comparative susceptibility corroborate this result, further experiments in the laboratory of various grapevine cultivars to the powdery mildew fungus with different sets of isolates from both groups are needed. Uncinula necator. Am. J. Enol. Vitic. 36, 101-104. If confirmed, differences in pathogenicity between genetic DOSTER, M. A.; SCHNATHORST, W. C.; 1985 b: Effects of leaf maturity and cultivar resistance on development of the powdery mildew fun- groups could contribute to changes in the global popula- gus on grapevines. Phytopathology 75, 318-321. tion with respect to their relative frequency. Therefore, lower FICKE, A.; GADOURY, D. M.; SEEM, R. C.; 2002: Ontogenic resistance and pathogenicity in group A than in group B could be another plant disease management: A case study of grape powdery mil- explanation for the shift that was observed during the grow- dew. Phytopathology 92, 671-675. FICKE, A.; GADOURY, D. M.; SEEM, R. C.; DRY, I. B.; 2003: Effects of ing season from a population consisting of group A isolates ontogenic resistance upon establishment and growth of Uncinula to a population consisting of group B isolates in necator on grape berries. Phytopathology 93, 556-563. (DÉLYE et al. 1999). FICKE, A.; GADOURY, D. M.; SEEM, R. C.; GODFREY, D.; DRY, I. B.; 2004: In conclusion, the method presented here has several Hosts barriers and responses to Uncinula necator in developing grape berries. Phytopathology 94, 438-445. potential applications such as cultivar assessment, analysis FISCHER, B. M.; SLAKHUTDINOV, I.; AKKURT, M.; EIBACH, R.; EDWARDS, K. J.; of host-specialization and studies on the variation in patho- TÖPFER, R.; ZYPRIAN, E.; 2004: Quantitative trait locus analysis of genicity in the fungus. Although the detached leaf assay fungal disease resistance factors on a molecular map of grape- may not fully reproduce the host-pathogen relationships in vine. Theor. Appl. Genet. 108, 501-515. GADOURY, D. M.; PEARSON, R. C.; 1991: Heterothallism and pathogenic the vineyard, we would expect the significant differences specialization in Uncinula necator. Phytopathology 81, evidenced between cultivars or isolates for only one infec- 1287-1293. tious cycle in a Petri plate to be amplified in the vineyard due GADOURY, D. M.; SEEM, R. C.; FICKE, A.; WILCOX, W. F.; 2003: Ontogenic to the polycyclic development of powdery mildew during resistance to powdery mildew in grape berries. Phytopathology 93, 547-555. the growing season. GADOURY, D. M.; SEEM, R. C., PEARSON, R. C.; WILCOX, W. F.; DUNST, R. M.; 2001: Effects of powdery mildew on growth, yield and quality of Concord . Plant Dis. 85, 137-140. Acknowledgements GALET, P.; 1988: Cépages et Vignobles de France. 1. Les Vignes Américaines, 2nd ed. Imprimerie Déhan, Montpellier. This work was partially supported by the “Comité Tech- KITAO, Y.; DOAZAN, J. P.; 1990: Grapevine breeding for resistance to nique Permanent de la Sélection” (grant No. C0047). We thank powdery mildew: Bioassay system for evaluation of plant resist- ance and for characterization of different Uncinula necator Dr. A. BOUQUET et Dr. A. DOLIGEZ for critical reading of the manu- th script. strains. Proc. 5 Inter. Symp. Grape Breed., 12-16 Sept.1989, St. Martin-Pfalz., Germany. Vitis (special issue), 249-253. LI, H.; 1993: Studies on the resistance of grapevine to powdery mil- References dew. Plant Pathol. 42, 792-796. PAUQUET, J.; BOUQUET, A.; THIS, P.; ADAM-BLONDON, A.-F.; 2001: Estab- lishment of a local map of AFLP markers around the powdery ANTCLIFF, A. C.; 1992: : The Grapevine as a Member of the mildew resistance gene Run1 in grapevine and assessment of Plant Kingdom. In: B. C. COOMBE, P. R. DRY (Eds): , their usefulness for Marker Assisted Selection. Theor. Appl. 107-118. Winetitles, Adelaide, Australia. Genet. 103, 1201-1210. BOUBALS, D.; 1961: Etude des causes de la résistance des Vitacées à PEARSON, R. C.; 1988: Powdery Mildew. In: R. C. PEARSON, A. C. GOHEEN l’oïdium de la vigne et de leur mode de transmission héréditaire. (Eds): Compendium of Grape Diseases, 9-10. Am. Phytopathol. Ann. Amélior. Plant. 11, 401-500. Soc., St Paul, USA. BOUQUET, A.; 1986: Introduction dans l’espèce Vitis vinifera L. d’un PÉROS, J. P.; TORREGROSA, L.; BERGER, G.; 1998: Variability among Vitis caractère de résistance à l’ (Uncinula necator Schw. Burr.) vinifera cultivars in micropropagation, organogenesis and anti- issu de l’espèce Muscadinia rotundifolia (Michx.) small. Vinevini biotic sensitivity. J. Exp. Bot. 49, 171-179. 12 (suppl), 141-146. PÉROS, J. P.; TROULET, C.; GUERRIERO, M.; MICHEL-ROMITI, C.; NOTTEGHEM, J. BRAUN, U.; TAKAMATSU, S.; 2000: Phylogeny of Erysiphe, Microsphaera, L.; 2005. Genetic variation and population structure of the grape Uncinula (Erysipheae) and Cystotheca, Podosphaera, Sphaero- powdery mildew fungus, Erysiphe necator, in southern France. theca (Cystotheceae) inferred from rDNA ITS sequences - some Eur. J. Plant Pathol. (in press). taxonomic consequences. Schlechtendalia 4, 1-33. STAUDT, G.; 1997: Evaluation of resistance to grapevine powdery mil- BULIT, J.; LAFON, R.; 1978: Powdery Mildew of the Vine. In: D. M. dew (Uncinula necator [Schw.]Burr., anamorph Oidium tuckeri SPENCER (Ed.): The Powdery Mildews, 525-548. Academic Press, Berk.) in accessions of Vitis species. Vitis 36, 151-154. New York, USA. STEIN, U.; HEINTZ, C.; BLAICH, R.; 1985: The in vitro examination of CALONNEC, A.; CARTOLARO, P.; POUPOT, C.; DUBOURDIEU, D.; DARRIET, P.; grapevines regarding resistance to powdery and . 2004: Effects of Uncinula necator on the yield and quality of J. Plant Dis. Prot. 92, 355-369. grapes (Vitis vinifera) and wine. Plant Pathol. 53, 434-445. STUMMER, B. E.; ZANKER T.; SCOTT E. S.; WHISSON D. L.; 2000: Genetic CHELLEMI, D. O.; MAROIS, J. J.; 1991: Sporulation of Uncinula necator diversity in populations of Uncinula necator: Comparison of on grape leaves as influenced by temperature and cultivar. Phy- RFLP- and PCR-based approaches. Mycol. Res. 104, 44-52. topathology 81, 197-201. STUMMER, B. E.; FRANCIS, I. L.; ZANKER T.; LATTEY, K. A.; SCOTT E. S.; COUTINHO, M. P.; MARTINS, A.; 1985: Quelques résultats sur la résistance 2005: Effects of powdery mildew on the sensory properties and de la vigne au mildiou et à l’oidium. C. R. 4éme Symp. Intern. Gen. composition of Chardonnay juice and wine when grape sugar Vigne, 13-18 April 1985, Verona, Italy. Vignevini 13, 148-152. ripeness is standardized. Aust. J. Grape Wine Res. 11, 66-76. DELP, C. J.; 1954: Effect of temperature and humidity on the grape WANG, Y.; LIU, Y.; HE, P.; CHEN, J.; LAMIKANRA, O.; LU, J.; 1995: Evalu- powdery mildew fungus. Phytopathology 44, 615-626. ation of foliar resistance to Uncinula necator in Chinese wild DÉLYE, C.; LAIGRET, F.; CORIO-COSTET, M. F.; 1997: RAPD analysis pro- Vitis species. Vitis 34, 159-164. vides insight into the biology and epidemiology of Uncinula necator 87 . Phytopathology , 670-677. Received June 23, 2005